携带RA538的重组体腺病毒体外及体内安全性研究

In vitro and in vivo evaluation of the safety of Ad-RA538

研究携带RA5 3 8的重组腺病毒Ad RA5 3 8潜在的副作用 ,评价其安全性 ,为其进一步进入临床试验提供依据。方法 体外病毒扩增实验 :预先用Ad RA5 3 8感染HeLa细胞 ,制备细胞提取液后反复两次感染HeLa细胞 ,观察Ad RA5 3 8的繁殖、扩增以及对HeLa细胞的作用 ,逆转录 聚合酶链反应 (RT PCR)检测其DNA的转录 ;确定Ad RA5 3 8对作为正常细胞的人胚肺二倍体细胞 2BS的感染能力 ,绘制细胞生长曲线 ,观察Ad RA5 3 8对正常细胞的毒性 ;给BalB/C小鼠腹腔注射重组腺病毒后 ,每日观察一般状况 ,化验肝、肾功能 ,PCR检测重组腺病毒在重要脏器中的分布 ,显微镜观察重要脏器的病理学变化。结果 Ad RA5 3 8是一复制缺陷型腺病毒 ,能高效感染 2BS细胞 ,但并不影响其生长 ,小鼠体内应用后 ,并无急性毒性症状发生 ,肝、肾功能没有变化 ,在肝、肾、胃、脾内可检测到腺病毒的分布 ,但仅在注射 10 9pfu第 6天、第 12天的小鼠肝组织内可观察到轻微炎症。结论 Ad RA5 3 8应用是安全的 ,可以进入临床试验。

Objective To assess the safety of adenovirus mediated transfer of the RA538 (Ad RA538) for the treatment of cancer and to furthermore in preparation for a clinical trial of Ad RA538 Methods RT PCR was used to detect the transcription of Ad RA538 in HeLa cells infected with extracts from HeLa cells previously infected with Ad5 RA538 Cell counting was made to observe the effects of Ad RA538 on the growth of the normal human fetal lung cell line 2BS The virus was intraperitoneally injected into 2 groups of BalB/C mice at a dosage of 10 7 pfu and 10 9 pfu Blood samples were taken from the mice to test the liver and renal function PCR were used to screen the vital organs for the presence of adenovirus DNA Microscopic examination of the vital organs was performed to observe the pathogenicity of Ad RA538 Results Ad RA538 was a replication defective virus It could infect 2BS cells effectively, but could not inhibit 2BS cell growth No mouse died and no signs of general toxicity were seen following intraperitoneal injection of Ad RA538 The adenoviral vector was present in the liver, spleen, kidney and stomach of mice injected with 10 9pfu Ad RA538 Six and 12 days after injection, mild inflammation was observed in the liver of mice received 10 9 pfu Ad RA538 Conclusion Ad RA538 is safe both in vivo and in vitro, and clinical trials of Ad RA538 can be performed